We constructed self-cloning diploid sake yeast strains that accumulate proline. The appropriate proline level is important for its protective effect against ethanol stress in yeast cells. Sake brewed with the proline-accumulating strains contained two- to threefold more proline than the sake brewed with the parent strain. It was also suggested that intracellular proline does not affect overall fermentation profiles, but reduces fermentation time in terms of ethanol production rate.